The detection of AChE inhibitors has been conducted using colorimetric methods that are based on decrease in color development by a primary or secondary substrate after exposure of the enzyme to a liquid sample containing an inhibitor. An example of such is the colorimetric method commercialized by Abraxis which uses Ellman's color reagent as an indicator substrate. That method tests the activity of a measured amount of enzyme after it has been exposed to a water sample potentially containing an inhibitor. After such exposure, the enzyme is reacted with its primary substrate acetylthiocholine. Active enzyme will hydrolyze acetylthiocholine to thiocholine which in turn reacts with dithio-bis-nitrobenzoate to produce an orange-colored product, 5-thio-2-nitrobenzoate.
That method, though widely used, is complex, a standard curve is needed, numerous fluid transfer pipetting steps are necessary, and a multiplicity of reaction vessels are used. In addition, the enzyme is inhibited by many organophosphates in vivo only after the inhibitor undergoes a physiological oxidation (Mahajna and Casida, 1998). Hence, a sample pretreatment step using an oxidizing agent, such as, bromine or hypochlorite followed by a quencher to neutralize excess unreacted oxidizer is required. Only by pre-oxidizing the sample can most organophosphates be detected by such methods.
Although such assays represent the current standard in AChE inhibitor detection, Ellman's Reagent-based methods are not converted readily to rapid on-site testing because of the methodological complexity, the numerous steps and reaction vessels required and the need to keep heat-labile reagents refrigerated.
Other methods to detect such inhibitors are laboratory-based liquid or gas chromatography methods coupled with mass spectrometry which rely on elution profiles for identification.
Rapid methods are also required to determine whether an individual has been exposed to neurotoxins to administer countermeasures and antidotes in timely fashion. Occupational exposure to pesticides, nerve agents and other cholinesterase-inhibiting metals is a common risk to individuals employed in the chemical, agricultural and mining industries and in the armed forces. Inhalation and absorption through unprotected skin are the most common modes of exposure.
Current methods for determining exposure consist of laboratory procedures based on the use of Ellman's Reagent. Approved methods include the Michel, microMichel, pH stat, Ellman and micro-Ellman assays, as well as certain variations of those assays.
Hence, there is a need for a simplified and rapid point of use assay. The instant invention addresses those shortcomings. The assay of interest can be configured to test plasma esterase, such as, cholinesterase, activity using, for example, plasma or serum obtained via venipuncture or diluted fingerstick blood, or environmental samples.